The incidence of tuberculosis (TB) has increased among the Veterans in recent years because the global burden of TB is enormous. This burden has escalated with the emergence of multidrug-resistant and extremely drug resistant Mycobacterium tuberculosis (Mtb) strains and because current vaccines do not elicit long-lasting protective immunity against TB especially in adults. Hence, the development of new generations of vaccines that will confer durable protection against TB will significantly improve the quality of life of our Veterans. Our plan proposes pre-clinical studies that will identify protective CD8+ T cell epitopes and develop mucosal vaccine delivery platforms for the design of next generation TB vaccines. Mtb enters the host through the respiratory tract. Hence, optimal protection will require lung-resident CD4+ and CD8+ memory T cells to be positioned at the frontline to respond immediately to infection. Traditional vaccines and approved adjuvants typically elicit weak, short-lived T cell responses, and parenteral vaccination is ineffective at installing protective immunity within tissue mucosae. Moreover, most virus-vectored and subunit TB vaccines employ a small subset of Mtb antigens, resulting in insufficient epitope diversity for optimal protection, partly because the epitopes that are presented during Mtb infection and confer protective immunity have not been explored. Hence, our overall objective is to discover immunogenic Mtb epitopes generated during infection and to incorporate them in an innovative nanoparticle (NP)-based intranasal vaccine that is designed to promote a balanced pulmonary CD4+ and CD8+ T cell responses that will protect against TB. In preliminary experiments, we have identified 41 novel peptides from Mtb H37Rv-infected primary macrophages using a proteomics approach. Among these, 17 are putative HLA-B*07;02-binding epitopes, which we will characterize to advance anti-TB vaccine design. Eliciting CD8+ T cells that complement a CD4+ T cell response requires that subunit antigens be presented by HLA class I molecules for CD8+ T cell cross-priming in the context of appropriate inflammatory cues that drive both CD8+ T cell and CD4 differentiation. Our team has recently pioneered a ?pathogen-mimicking? vaccine that is based on pH-responsive, endosome-rupturing NP chemistry. Such NPs promote delivery of antigens and nucleic acid adjuvant cargoes into the cytooplasm. Preliminary studies demonstrated that a single intranasal administration of NPs loaded with ovalbumin in conjunction with a structurally optimized 5'ppp-RNA hairpin adjuvant, that activates cytoplasmic retinoic acid-inducible gene-I, elicits a robust, durable and protective antigen-specific T cell response in the lungs. Based on these exciting new findings, we hypothesize that intranasal immunization with NP vaccines co-loaded with naturally processed class I-restricted Mtb-derived epitopes and 5'ppp-RNA adjuvant will significantly enhance tissue resident CD8+ memory T cell responses in the airways and parenchyma of the lungs. Our strategy to test this hypothesis is to, (a) evaluate the immunogenicity and protective potential of naturally processed Mtb epitopes presented by HLA-B*07:02 class I molecules; (b) determine whether HLA-B*07:02 and B*35:01?two closely-related HLA molecules?present similar epitopes; and (c) develop a NP vaccine that elicits a durable lung-resident CD8+ and CD4+ memory T cell responses to Mtb subunit antigen(s) that confers protection against TB. Our multidisciplinary team ?consisting of biochemists, immunologists, microbiologists and bio-engineer? is ideally situated to pursuing the three Specific Aims. We anticipate that successful completion of the proposed research will yield highly significant knowledge essential for next generation anti-TB vaccine design. We expect to discover novel protective Mtb epitopes presented and recognized by multiple HLA molecules. We will also have developed a new delivery platform for mucosal vaccination that will allow co-delivery of antigens and adjuvants for cytoplasmic immune surveillance. Our innovative ?discover and deliver? approach to vaccine design, will impact clinical practice paradigms against TB and other intranasal infections and, hence, will better the healthcare of our Veterans.